Chromatographic column head space reducer



9 19m .1, A. MWmE-mm CHROMATOGRAPHIC COLUMN HEAD SPACE EEDUCER 2Sheets-Sheet 1 Filed Aug. 5, 1968 N w OR- T m N E w T mm P J A W S E M mMl EATTORNEYS mmw 1970 J A. PATTEEIWSEOM 5F7V93 CHROMATOGRAPHIC COLUMNHEAD SPACE HEDUCER Filed Aug. 8, 1968 2 Sheets-Sheet 2 BUFFER FLOWSAMPLE BNJECTOR FILTER IN VENTOR.

I JAMES A. PATTERSON BY 56-. 5 "70 m! WW ATFORNEYS United States Patent3,487,938 CHROMATOGRAPHIC COLUMN HEAD SPACE REDUCER James A. Patterson,Los Altos, Califi, assignor t9 Sondell Research & Development Co., PaloAlto, Callf., a corporation of California Filed Aug. 8, 1968, Ser. No.752,103

Int. Cl. B01d /08 US. Cl. 210-198 15 Claims ABSTRACT OF THE DISCLOSUREAn automatic head space reducer for chromatographic columns is formedfrom cylinder means positioned over and fixed relative to thechromatographic column for receiving fluid under pressure from the fluidline, and piston means positioned within the head space of thechromatographic column slidably engaging the cylinder means. The pistonis slidable within the head space, in response to fluid pressuretransmitted through the fluid line and cylinder means, to minimize thehead space and rest with a predetermined minimal differential forceagainst chromatographic filter material retained in the column, whiledelivering fluid to the filter material from the fluid line through achannel in the piston.

This invention relates to a new and improved automatic head spacereducer for chromatographic columns for eliminating the head space abovechromatographic filter material retained in a chromatographic columnthrough which the sample must be introduced.

Chromatographic columns generally comprise an elongated cylindricalhousing having chromatographic filter material retained therein and ahead space within the housing above the filter material through which asample must be introduced. In order to provide a sharp chromatogram ofthe sample it is necessary that the sample be initiated into the columnof chromatographic filter material in as narrow a zone as possible. Itis thus desirable that the entire sample he introduced simultaneouslyacross the top of the column of filter material. If there is any freespace or head space above the filter material during loading of asample, a concentration gradient, pH gradient or other gradient may beestablished across the bufler solution in the head space. The resultingdilution of the sample results in a delayed introduction of the sampleinto the filter material thereby destroying the sharpness of theresulting chromatogram. Eflicient and high resolution columnchromatography thus requires that the head space above the column offilter material be minimized.

According to present techniques for minimizing the head space above thefilter material in a chromatographic column, porous plugs or headspacers are manually positioned in the head space. Such manual headspacers may be screw-adjusted or otherwise manually adjusted against thetop of the column of chromatographic filter material. By such manualplacement, an unknown amount of physical pressure is placed against thefilter material. Chromatographic filter materials such as cross-linkedpolymer resin beads are sensitive to such applied pressure. Thus, theosmotic or absorptive activity of the surface of the resin or otherfilter material may be adversely afiected by the pressure and therebyinterfere with the sharpness and resolution of the resultingchromatograph. Furthermore, such manual placement may create pressuresbeyond the rupture limit of the system.

During gradient elution or elution of successive samples of varyingconcentration in chromatographic columns of the type using copolymerresin beads as a chromatographic filter material, the resin beads expandand contract,

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changing volume under the varying solvation stresses to which the beadsare subjected. In systems wherein a porous plug is manually positionedin the column head space, the change in volume of the resin beads underthe plug may subject the resin beads to physical pressures which destroyresolution of the chromatogram or which exceed the rupture limit of thesystem. To correct for the changing volume of the resin beads due tochanging solvation stresses, the plug must be continua-11y manuallyadjusted, and even then with only limited accuracy.

It is an object of the present invention to provide a new and improvedautomatic head space reducer for minimizing head space inchromatographic columns by the application of predetermined andcontrolled pressures, thereby avoiding application of adverse or unknownphysical pressure to the filter material or creation of harmfulpressures in the fluid system.

Another object of the invention is to provide an automatic head spacereducer which displaces head space in chromatographic columns byproviding a movable piston which rests on the column of chromatographicfilter material with a predetermined minimal differential force andwhich automatically follows the filter bed with the predeterminedminimal differential force during change of volume of the column ofchromatographic filter material.

A further object of the invention is to utilize the pressure in thefluid buffer line of the chromatographic system for automaticallydisplacing the free head space above the filter material.

In order to accomplish these results the present invention contemplatesproviding a cylinder or pressure chamber positioned over and fixedrelative to the chromatographic column. The cylinder or chamber includesan inlet for receiving fluid under pressure, an outlet to expel receivedfluid, and at least one channel for receiving and actuating a piston.One end of a piston is positioned slidably within the cylinder while theother end is positioned within the head space of the chromatographiccolumn above the filter material and slides within the column todisplace the head space in response to fluid pressure in the cylinder.

According to one aspect of the invention, the sample fluid and bufiersolution are fed under pressure into the cylinder. The resultingpressure drives the piston downward to displace the head space over thechromatographic filter material. Upon leaving the cylinder, the fluidenters a passageway in the piston, applying pressure on a pressurerelease valve also formed in the piston. When the piston is urgedagainst the chromatographic filter material, the valve opens at apredetermined differential force to release the sample and buifer at thehead of the column of chromatographic filter material. The pressureexerted on the filter material is thereby controlled to a minimal level.Pressure from the sample and butfer line is utilized to displace thepiston into the head space, the sample and buffer subsequently beingreleased through the piston onto the chromatographic filter materialupon development of a predetermined minimal differential force downwardagainst the piston pressure release valve.

According to another aspect of the invention the cylinder comprises aseparate pressure chamber having an inlet and outlet for receiving anddischarging bulfer fluid under pressure from the fluid line for drivingthe piston. The piston is designed to rest with a predetermined minimaldifferential force against the chromatographic filter material despitechanges in back pressure and changes in volume of the filter material.The operative differential force is the difference between the forcedownward on the piston from the pressure in sample and buffer fluid lineand the force upward due to pressure upward on the piston from thechromatographic column. The piston is provided with a separatelongitudinal passageway therethrough for receiving and delivering thesample fluid received from a sample injector and butter solution fromthe fluid line to the chromatographic column. The walls of the housingfor the chromatographic column may be utilized to form part of thepressure chamber. In each case the piston is provided with an O-ringseal for sealing engagement with the walls of the housing of thechromatographic column. The invention also contemplates a piston designwhich absorbs the shock or pulses introduced into the fluid line by apositive displacement pump without transmitting the pressure pulses tothe filter material in the chromatographic column.

Other objects, features and advantages of the present invention willbecome apparent in the following specification and accompanyingdrawings.

FIG. 1 is a side cross-sectional view of an automatic head space reducerembodying the present invention with the piston and head spacerdisassembled.

FIG. 2 is a side cross-sectional view of another form of automatic headspace reducer embodying the present invention with the piston and headspacer disassembled.

FIG. 3 is a side cross-sectional view of another automatic head spacereducer embodying the present in vention.

FIG. 4 is a block diagram of the fluid system for a chromatographiccolumn and any of the automatic head space reducers illustrated in FIGS.1 through 3.

FIG. 5 is a side view of a form of automatic head space reducerembodying the present invention in which the sample and buffer solutionare used to drive the piston directly.

FIG. 6 is a disassembled side cross-sectional view of the automatic headspace reducer illustrated in FIG. 5. FIG. 6a is a side cross-sectionalview of the piston.

In the embodiment of the present invention illustrated in FIG. 1 thereis provided a cylinder 10, providing a pressure chamber, fixed to amounting 11 positioned over and stationary relative to a chromatographiccolumn 12. The chromatographic column is formed by way of example froman elongated cylindrical housing 13 having a chromatographic filtermaterial 14 such as a multitude of cross-linked co-polymer resin beadsretained therein by a screen 15 at each end of the column of resinbeads. The screen 15 may be a micron mesh stainless steel screen forretaining the resin beads. Such a chromatographic column is described inmy US. patent application Ser. No. 530,051, entitled Microsphere Filterand Method of Filtration filed on Feb. 25, 1966. The invention isapplicable, however, to other types of chromatographic columns.

Slidably mounted through channels 16 and 17 formed through oppositesides of the cylinder 10 respectively, is a piston 18, having O-ringseals 20 and 21 for engaging the channels 16 and 17, respectively, ofthe cylinder 10. The O-ring seals are adapted to slide within thechannels while maintaining a sealing relationship between the piston 18and cylinder 10. The portion of piston 18 within the pressure chamber 22defined by the cylinder housing 10 is provided with differentialdiameters D and D The upper portion of piston 18 within the pressurechamber 22 of diameter D is of narrower diameter than the lower portionof the piston within chamber 22 having a diameter of D so that a netdownward force is exerted on the piston 18 when fluid under pressure isadmitted into the pressure chamber 22 through an inlet 23 formed in thecylinder for receiving fluid from a pump. An outlet 24 is also providedthrough the cylinder wall from the pressure chamber 22 for ejecting thefluid under pressure.

The piston 18 is provided with a detachable extension 26 which serves asthe head spacer for the chromatographic column. The head spacer 26 maybe attached to the piston 18 by complementary threaded screws 27provided on an extension and a recess in the head spacer 26 and piston18, respectively. An O-ring seal 28 is provided in the recess to providea fluid seal between the threaded portions for reasons hereinafterdescribed. The extension 26 which for-ms the head space reducer isformed of dimensions complementary to and slightly smaller than the headspace 30 formed in the chromatographic column over the filter material14. The head spacer 26 is provided with an O-ring seal 31 which slidablyengages the inner wall of the housing 13 for the chromatographic columnin sealing relationship. The head spacer 26 which is attached to thepiston 18 may be any of a variety of sizes chosen to displace the headspace in the chromatographic column being used.

An axial passageway 34 is provided down the center of piston 18 and theattached head spacer 26 including an inlet 35 in the piston forreceiving a sample and buffer solution and an outlet 36 in the headspacer for depositing the sample and buffer solution conducted downpassageway 34 onto the top of the bed of resin beads 14 or otherchromatographic filter material.

In operating the automatic head space reducer, the head spacer 26 isscrewed into the piston 18 and positioned within the head space 30 of achromatographic colum. The inlet 35 to passageway 34 within the piston18 is opened to permit a buffer solution to fill the open head spacebetween the top of the column of resin beads 14 and the head spacer 26.The mountings 11 in cylinder 10' are then positioned down on the pistonagainst the bottom stops 36- formed on the piston and secured withrespect to the chromatographic column so that the cylinder is as fardown on the piston as possible. A buffer solution under pressure is thenadmitted through the inlet 23 into pressure chamber 22 of the cylinder10 so that a downward force which is a function of the fluid pressureand the differential area between the portions of the piston of diameterD and D is applied to the piston. The differential area on piston 18within the pressure chamber 22 is chosen so that the resultant forceslightly exceeds the upward force on the piston from the back pressurefrom the chromatographic column and the frictional force of the O-ringseals 20, 21 and 31 so that the piston and head spacer slide downward torest on the screen 15 and filter material 14 with a predeterminedminimal force. A retainer ring 19* is provided around the top of thepiston to prevent the piston from travelling downward further than apredetermined distance.

Referring to FIG. 4, by way of example, butter solution is applied tothe fluid line at a predetermined pressure by the pump 40. A filter 41in the fluid line filters the buffer solution which then enters thepressure chamber 42 within the cylinder. From the pressure chamber 42,the fluid is released into a sample injector 43 which provides a knownresistance in the fluid line injecting the sample into the buffersolution. The injected sample and buffer solution are then conductedthrough the center of the piston and head spacer 44 to thechromatographic column and out the output end of the fluid line.

Thus, if a pump pressure of P is provided the upward force F due to backpressure from the chromatographic column 45 on the piston and headspacer 44 is given by the following equation where D is the diameter ofthe head spacer 26 at the end adjacent the chromatographic filtermaterial.

The downward force F on the piston and head spacer 44 from pressurechamber 42 is given by the following equation where D and D are thedifferential diameters, respectively, of the portion of the pistonwithin the pressure chamber 22 and R is the resistive pressure providedin the fluid line by the sample injector 43 which resistive pressure maybe controlled to equal zero.

The downward force F on the piston and head spacer 44 must be chosen toslightly exceed the upward force F and the frictional resistance of theO-ring seals on the piston and head spacer so that the head spacer comesto rest on the column of resin beads or other filter material with apredetermined minimal force.

The materials of the automatic head space reducer must be chosen so thatthe cylinder and piston and head spacer and column have compatiblecoefficients of thermal expansion.

In the embodiment of the invention illustrated in FIG. 2 an automatichead spacer reducer is shown wherein frictional forces from the O-ring.seals are minimized by reducing the area of O-ring seal contact betweenthe piston and cylinder and the head spacer and chromatographic column.As shown in FIG. 2, the piston 50 is of reduced diameter and extendsthrough a single channel 51 in a cylinder 52 which defines a pressurechamber 53. The piston 50 terminates within the pressure chamber with atop pressure surface 54 including a depending diaphragm 55 extendingbetween the perimeter of the pressure surface 54 and the bottom of thecylinder 52. The piston is adapted to slide within the channel 51 insealing engagement with O-ring seal 56 upon application of pressure tothe top pressure surface 54. Inlet 57 and outlet 58 are provided in thecylinder communicating with the pressure chamber 53 for receiving anddischarging a fluid under pressure for transmitting pressure to the toppressure surface 54. Attached to the end of the piston 50 outside thepressure chamber is a connector 60 which is in turn connected to a headspacer 61 which may be of variable size for displacement of head spacein the chromatographic column with which it is to be used. The piston50, connector 60 and head spacer 61 may be connected by suitablethreaded extensions and recesses. The connector 60 includes an inlet 62for receiving a buffer solution and sample for delivery through theaxial channel 63 formed down the center of the connector 60 and headspacer 61. An O-ring seal 64 is provided in the threaded recess 65 inthe connector 60 for sealing engagement between the connector and headspacer 61 to provide a continuous channel 63. The buffer solution andsample may then be delivered through the channel 63 to the head space 66over the column of chromatographic material 67 in the chromatographiccolumn 68 which the head spacer 61 is seated. The head spacer includesan O-ring 70 to provide sealing engagement between the head spacer 61and the wall of the chromatographic column 68.

The automatic head space reducer illustrated in FIG. 2 may beincorporated in a fluid circuit such as illustrated in FIG. 4. The areaof top pressure surface 54 is chosen to provide a downward force on thepiston from pressure in the fluid line, which exceeds the upward forcefrom the back pressure on the head spacer 61 from the chromatographiccolumn and the' frictional force of the O-ring seals 51 and 70 so thatthe head spacer comes to rest upon the top of the column ofchromatographic filter material with a predetermined minimal force.Because the contact area between the O-ring seals and sliding surfaceshas been reduced, a smaller differential force may be used and there isless likelihood of surging. The automatic head space reducer is operatedin the manner heretofore described with the cylinder 52 mounted over andin alignment with the chromatographic column 68.

In the head space reducer illustrated in FIG. 3, the walls 80 of achromatographic column 81 provide the side walls for the pressurechamber 82 in which fluid under pressure is received. The top of thepressure chamher comprises a stopper 83 placed in the head of thechromatographic column and in sealing engagement therewith by means ofO-ring seal 84. The stopper 83 and walls thus provide a cylinder whichdefines the pressure chamber 82. The stopper 83 includes an inlet 85 forreceiving fluid under pressure and an outlet 86 for rejecting the fluidand is mounted stationary relative to the chromatographic columns bymeans of a mounting frame 87. A piston 88 positioned in the head spaceof the chromatographic column over the chromatographic filter material90 is provided with a narrow shaft 91 slidably extending through achannel 92 provided centrally through the stopper 83. O-ring seals 93and 94 provide a sealing engagement between the stopper 83 and shaft 91.The piston 88 and shaft 91 include an axial passageway 95 longitudinaldown the center of the shaft and piston and an inlet 96 for receivingbuffer solution and sample fluid and delivering the butter and samplethrough the passageway 95 and out the opposite end 97 of the channel 95to the column of chromatographic filter material 90.

The piston 88 is provided with tapered surfaces 98 and 100, taperinginwardly from larger diameters at the ends to a central recess ofsmaller diameter where a large O-ring seal 101 is seated. With theO-ring 101 centered between the tapers 98 and 100, a force downward onthe head surface 102 of piston 88 and O-ring 101 slightly greater thanthe upward force due to back pressure from the chromatographic column onthe bottom surface 103 will push the piston downward against the columnof chromatographic material to rest thereon with predetermined minimalforce. The O-ring seal 101 will slide easily along the wall 80 of thechromatographic column 81 under the slight difierential pressure as longas it is seated in the recess centered between the tapered sides 98 andof the piston 88. However, under violent pressure changes and pressuresurge from a displacement pump in the fluid line, the O-ring seal 101will lodge between the wall 80 of the chromatographic column and eitherof the tapered surfaces 98 and 100 to restrict movement of the piston 88and thereby act as a shock absorber to absorb surging pressures in thefluid line from the displacement pump or other sources. The hammereffect on the resin surface due to pump variation will therefore beminimized.

The automatic head space reducer illustrated in FIG. 3 will tolerate agreater differential coefiicient thermal expansion between the headspacer piston 88 and the chromatographic column than in the previousembodiments because the O-ring seal is larger and is not held in a fixedslot. On the other hand, the larger O-ring seal 101 does not increasethe friction because under the normal ditferential pressure frompressure chamber 82 the O-ring seal 101 is centered between the taperedsurfaces 98 and 100 so that it slides easily within the walls of thechromatographic column. Thus, when pressure is applied to the fluid lineby a buffer solution pump, the head spacer piston 88 will come to reston the column of chromatographic filter material with a predeterminedminimal force and follow the column of filter material downwardly uponcompaction of the filter material under pressure and upwardly uponexpansion of the filter material thereby continually eliminating thehead space and controlling the pressure on the filter material.

In operation of each of the head space reducers illustrated in FIGURES 1through 3, a priming butter solution is first fed down the central axialchannel within the piston to fill the head space above thechromatographic column so that the fluid line is primed before thebuffer solution pump is turned on. With the pump turned off,difierential forces on the piston cause the piston to slide downwardfollowing the compacting column of filter material, continuouslyeliminating the head space.

FIGURES 5 and 6 illustrate an automatic head space reducer in which theneed for a pressure chamber upstream from and separate from the sampleinjector input to the chromatographic column is eliminated. In theembodiment of the invention illustrated in FIGURES and 6, fluid underpressure received in a buffer solution and sample inlet itself providesthe pressure for driving a piston to displace the head space within achromatographic column. As shown in those figures, a stopper or cylinder110 is provided over a chromatographic column on a mounting frame 111.The outside of the cylinder 110 is in sealing engagement with the Wall112 of the chromatographic column 113 by means of O-ring seal 114. Thecylinder 110 includes a longitudinal recess 115 extending axially fromthe bottom end within the chromatographic column and further includes atubular passageway 116 extending axially within the longitudinal recess115 and out the top of the cylinder to an inlet opening 117 forreceiving buffer solution and injected sample fluid under pressure. Thepiston comprises a piston head spacer 118 adapted to fit within the headspace of a chromatographic column and a tubular channel 120 extendingthrough the piston head 118 and upwardly therefrom. The tubular channel120 is adapted to axially enclose the tubular passageway 116 in slidingrelationship. The tubular channel extends within the longitudinal recess115 in the cylinder 110 and at its outer surface engages an O-ring seal121 in the longitudinal recess 115 to provide sealing engagement. Thelongitudinal recess 115 thereby defines a pressure chamber for exertinga downward force on the piston 118 via a tubular channel 120. Thetubular channel 120 terminates at the bottomend of the piston 118adjacent the chromatographic column in a ball valve comprised of a ball122 of metal or other suitable material seated within a complementaryorifice in the base of the piston. The ball 122 is retained underpressure against the channel 120 by a membrane 123 held against thebottom of the piston head 118 with a predetermined force. The membrane123 may form the bottom of a cap 124 to screw over the bottom of pistonhead 118 along threads provided thereon. The membrane 123 may be aplastic membrane or other suitable membrane having holes therein fordistributing fluid along the top of the column of chromatographic filtermaterial. The membrane 123 retains the ball 122 in position locking thechannel 120 with a predetermined force of, for example, a half pound.

In operating the automatic head space reducer shown in FIGURES 4 through6, the head space over a chromatographic column is first filled with abuffer solution andthe piston 118 is positioned in the head space withthe cylinder 110 mounted over the chromatographic column. A buffersolution under pressure is received in the inlet 117 through a fluidline 126 and fed to the tubular channel 120 of the piston 118.

Buffer solution under pressure is received in the inlet 117 from fluidline 126 and is fed through the tubular passageway 116 to the tubularchannel 120 so that a net downward force is applied to the piston 118.The tubular channel 120 slides downward within the longitudinaldepression 115 and the piston head 118 displaces buffer solution in thehead space in the column of chromatographic filter material, to come torest on the top of the column of chromatographic filter material with apredetermined minimal force. The pressure of the piston on thechromatographic filter material is determined by the release pressure ofthe ball valve comprising the ball 122 and membrane 123. The valve maybe adjusted to provide release of buffer solution and sample from thetubular channel 120 through the membrane 123 upon creation of adifferential force downward in the tubular channel 120 of, for example,a half pound. Thus, the piston 118 will come to rest on the column ofchromatographic filter material with a pressure of a half pound, theexcess pressure producing release of butter solution and injected sampleunder pressure through the tubular channel 120. The piston 118 whichdisplaces the head space at the top of the chromatographic column isthereby driven directly by the fluid pressure from the buffer solutionand injected sample inlet 117.

What I claim is:

1. An automatic head space reducer for a chromatographic columnhaving'an elongated housing, chromatographic filter material retainedwithin the housing, and head space within the housing over the filtermaterial and a fluid line for delivering buffer solution and samples tothe column comprising:

displacement means positioned in the head space of the chromatographiccolumn and slidably engaging the housing of the chromatographic column,said displacement means slidable in response to downward forces producedby pressure in the fluid line and upward forces produced by backpressure transmitted from the chromatographic column, said displacementmeans adapted to rest, in response to pressure from the fluid line, onthe chromatographic filter material with a downward force in excess ofupward force, due to back pressure, by a predetermined amount, saiddisplacement means having a channel for delivering fluid from the fluidline to the chromatographic filter material,

and means transmitting downward pressure from the fluid line to saiddisplacement means.

2. An automatic head space reducer for a chromatographic column havingan elongated housing, chromatographic filter material retained withinthe housing, head space within the housing over the filter material, anda fluid line for delivering buffer solution and samples to the columncomprising:

cylinder means positioned over and fixed relative to the chromatographiccolumn, said cylinder means having an inlet adapted to receive fluidunder pressure from the fluid line and an outlet adapted to expel fluidreceived under pressure;

and piston means positioned within the head space of the chromatographiccolumn and slidably engaging said cylinder means, said piston beingslidable within the head space of the chromatographic column in responseto fluid pressure transmitted through the fluid line and cylinder means,said piston means adapted to deliver fluid from the cylinder means tothe filter material through a channel formed through the piston, saidpiston means adapted to rest on the column of filter material with adownward force, due to pressure in the fluid line, in excess of theupward force, due to pressure on the piston, by a predetermined amount.

3. An automatic head space reducer as set forth in claim 2 wherein theoutlet from said cylinder means comprises an elongated tubularpassageway and wherein said piston comprises an elongated tubular inletaxially enclosing and slidable in sealing engagement, relatlve to theelongated tubular passageway from the cylinder means, and a piston headconnected to the tubular lnlet including an internal channel and anoutlet and valve means for releasing through said outlet to thechromatographic column, fluid received from said cylinder means having apressure in excess of a predetermined amount.

4. An automatic head space reducer as set forth in claim 3 wherein saidvalve comprises a ball seated in the outlet from said piston and amembrane retained under pressure against said ball and piston.

5. An automatic head space reducer as set forth in claim 2 wherein partof said cylinder means comprises the housing of the chromatographiccolumn, and wherein a stopper is provided at the top of thechromatographlc column housing having an inlet and outlet for receivingand delivering fluid under pressure from the fluid line, and a channelfor receiving said piston means in slidable relationship.

6. An automatic head space reducer as set forth in claim 5 wherein thepiston means includes a piston head and an O-ring seal for sealingengagement between the piston head and housing of the chromatographiccolumn, said piston head having sides tapering from maximum widths ateach end to a minimum width in the middle, said O-ring seal positionedon the piston substantially at the point of minimum width, said pistonmeans also including a passageway therethrough for receiving anddelivering a sample to the chromatographic cloumn.

7. An automatic head space reducer for a chromato graphic column havingan elongated housing, chromatographic filter material retained withinthe housing, head space within the housing over the filter material anda fluid line for delivering buffer solution and samples to the columncomprising:

a pressure chamber having an inlet for receiving fluid under pressurefrom the fluidline and an outlet for releasing received fluid into thefluid line, said pressure chamber being positioned over and fixedrelative to a chromatographic column, said pressure chamber having atleast one channel for receiving a piston;

elongated piston means positioned in the head space of thechromatographic column and communicating with said pressure chamberthrough the receiving channel, said piston means slidable within thehead space of the chromatographic column in response to fluid pressurein said pressure chamber, said piston means including a longitudinalpassageway therethrough for receiving and delivering a sample and fluidfrom the fluid line to the chromatographic col umn, said piston adaptedto rest with a predetermined minimal differential force on thechromatographic filter material in response to fluid pressure in thefluid line.

8. An automatic head space reducer as set forth in claim 7 wherein saidpiston means includes an O-ring seal for sealing engagement with theinside of said chromatographic column housing.

9. An automatic head space reducer as set forth in claim 7 wherein saidpiston means includes a plurality of attachable sections for adjustingthe length of said piston.

10. An automatic head space reducer as set forth in claim 7 wherein saidpressure chamber comprises one channel for receiving a piston and saidpiston comprises a relatively narrow section extending through saidchannel, a relatively wide pressure surface within said pressure chamberand a diaphram depending from the perimeter of said pressure surface toa wall of said pressure chamber.

11. An automatic head space reducer as set forth in claim 7 wherein saidpressure chamber includes two channels in alignment on opposite sidesfor. receiving a piston therethrough and said piston extends through thepressure chamber, said piston comprising sections of differentialdiameter within the pressure chamber adapted to provide a force downwardon the piston in excess of force upward, due to back pressure on thepiston, by a predetermined amount.

12. An automatic head space reducer as set forth in claim 7 wherein theside walls of said pressure chamber comprise the housing of thechromatographic column and said piston comprises a piston head slidablewithin the head space of the chromatographic column, said piston headincluding an O-ring seal for sealing engagement with the housing of thechromatographic column.

13. An automatic head space reducer system for chromatographic columnscomprising:

a fluid line including pump means for delivering fluid under pressure insaid line;

a pressure chamber having an inlet for receiving fluid under pressurefrom said line and an outlet for releasing received fluid and at leastone channel for receiving a piston means;

a chromatographic column comprising an elongated housing,chromatographic filter material retained in said housing, and head spacewithin said housing over the filter material;

elongated piston means positioned in the head space of thechromatographic column communicating with said pressure chamber throughthe receiving channel, said piston means slidable within the head spaceof the chromatographic colunm relative to the pressure chamber inresponse to fluid pressure in the pressure chamber, said piston meansincluding a longitudinal passageway therethrough having an inlet and anoutlet;

sample injector means having an inlet and an outlet;

said fluid line including means connecting the output from said pressurechamber to the inlet to said sample injector, and the outlet from saidsample injector to the inlet to said piston means whereby a sample maybe delivered through the outlet from said piston means to thechromatographic column,

said piston means adapted to rest on the chromatographic filter materialwith a predetermined differential force downward in response to fluidpressure in the fluid line and pressure chamber.

14 An automatic head space reducer for a chromatographic column havingan elongated housing, chromatographic filter material retained withinthe housing, and head space within the housing over the filter materialcomprising:

cylinder means positioned over and fixed relative to the chromatographiccolumn, said cylinder means comprising an elongated tubular passagewayhaving an inlet adapted to receive fluid under pressure and an outletadapted to expel fluid under pressure;

and piston means positioned within the head space of the chromatographiccolumn and slidably engaging said cylinder means, the piston meansslidable within the head space of the chromatographic column in responseto fluid pressure transmitted from said cylinder means, said pistoncomprising an elongated tubular inlet axially enclosing and slidablerelative to the elongated tubular passageway fro-m the cylinder means,and a piston head connected to the tubular inlet, said piston headhaving an internal channel and an outlet and valve means in said outletfor releasing through said channel and outlet to the chromatographiccolumn, fluid received from said cylinder when a predetermined pressuredifferential occurs across said valve means.

15. An automatic head space reducer as set forth in claim 14 whereinsaid valve comprises a ball seated in the outlet from said piston headand a membrane retained under pressure against said ball and pistonhead.

References Cited UNITED STATES PATENTS 1,453,310 5/1923 Engel 210-350 XJAMES L. DECESARE, Primary Examiner US. Cl. X.R. 55-386; 210-351

